The method relates to a novel method for preparing urethane-protected N-carboxyanhydrides of alpha amino acids. With the novel method, urethane-protected N-carboxyanhydrides of alpha amino acids may be synthesized from N-carboxyanhydrides of alpha amino acids in the presence of a catalytic amount of triethylene diamine, without adding significant amounts of a base of the tertiary amine type.
N-carboxyanhydrides of alpha amino acids (designated under the acronym of NCAs), optionally protected, are acylation agents often used for forming high molecular weight polyalpha-amino acids and for producing dipeptides. NCAs are very reactive compounds, which do not form, notably by rearrangement, any undesired secondary products and their unique reaction byproduct is carbon dioxide. As soon as NCA is reacted with a free amine function of an amino acid, carbon dioxide is immediately released and a dipeptide is formed, which itself also contains a free amine function. This amine will react with NCA and form a tripeptide and so forth. NCAs may thereby be used in forming poly(alpha amino acids) but they cannot easily be used in the sequential synthesis of polypeptides, as multiple condensation secondary reactions such as oligomerisation, are difficult to control.
Alpha amino acid N-carboxyanhydrides substituted with urethane groups have been described in the literature, they are used in peptide syntheses. The urethane substituent provides a high degree of protection and allows polymerisation reactions to be minimized during the coupling reaction. Urethane-protected NCAs, abbreviated as UNCAs hereafter, have all the advantages of non-substituted NCAs without the drawbacks of the latter.
UNCAs allow controlled synthesis of polypeptides without requiring any pre-activation of the carboxyl groups and without requiring any addition of additives such as N-hydroxybenzotriazole. Thus, purification of the peptides produced in solution is facilitated, since the only byproduct of the peptide synthesis reaction is carbon dioxide.
UNCAs are also very useful as raw materials in the synthesis of hormones or anti-AIDS drugs.
UNCAs which are in a crystalline form under room temperatures and pressure conditions, are stable under standard laboratory handling and storage conditions and under peptide synthesis conditions.
The main two routes for synthesis of UNCAs from NCAs are the following:
1) UNCAs may be synthetized by condensation of an alkyl or aralkyl chloroformate, such as Fmoc-Cl (9-fluorenylmethyloxycarbonyl chloroformate) or benzyl chloroformate, with an NCA in presence of at least a stoichiometric amount of a tertiary amine. This tertiary amine which conventionally is N-methylmorpholine, the released hydrochloric acid to scavenged. NCA is thereby put into solution in the inner solvent, such as THF, and cooled. 1.1 to 1.3 equivalents of alkyl or aralkyl chloroformate is added only once and then at least 1.5 equivalents of tertiary amine, for example N-methylmorpholine, is slowly added. The resulting suspension is left at rest for 1 to 2 hours, at a temperature between −25 and −5° C. Next, hydrochloric acid dissolved in dioxane is slowly added until pH values of about 4-5 are obtained. Then, formed hydrochloride of the tertiary amine is filtered off and UNCA is concentrated and crystallized.
All the steps of the method are performed under an inert atmosphere (N2) and all the solvents are dried on a 4 Å molecular sieve before being used (William D. Fuller et al., Urethane-protected-alpha-amino acid N-carboxyanhydrides and peptide synthesis, Biopolymers, 1996, 40, 183-205).
This synthesis route is not very suitable for preparing certain protected alpha amino acid N-carboxyanhydrides, notably those protected by a t-butoxylcarbonyl radical, t-butyl chloroformate being very unstable above −20° C. or in the presence of tertiary amines.
2) UNCAs may also be synthesized by condensation of a dialkyl dicarbonate with an NCA. This reaction releases an alcohol molecule and a carbon dioxide molecule. This synthesis must absolutely be carried out in the presence of a large amount, at least a 50% molar amount relatively to the engaged NCA molar amount, of a tertiary amine such as N-methylmorpholine associated with a catalytic amount of DMAP (4-dimethylamino-pyridine) or a pyridine (William D. Fuller et al., Urethane-protected-alpha-amino acid N-carboxyanhydrides and peptides synthesis, Biopolymers, 1996, 40, 183-205). This synthesis route is particularly suited for the synthesis of alpha-amino acid N-carboxyanhydrides protected by a t-butoxylcarbonyl radical by using di-tertiobutyl dicarbonate.
Application WO89/08643 describes N-carboxyanhydrides of alpha-amino acids and N-thiocarboxy-anhydrides of alpha-amino acids with urethane protection, of formula,
wherein R and R′ represent a hydrogen atom, an alkyl, cycloalkyl, cycloalkyl radical substituted with a substituted alkyl, aryl or substituted aryl radical, and at least one R or R′ group does not represent a hydrogen atom; R″ represents an alkyl, aryl, substituted alkyl or substituted aryl; Z represents an oxygen or sulphur atom and n is 0, 1 or 2.
These compounds are prepared by reaction of NCA with a haloformate in an inert solvent, such as toluene, under anhydrous conditions, in the presence of an tertiary amine type base added in excess.
The existing methods for synthesizing UNCAs are not satisfactory. Actually, the best method described above, which uses a base of the tertiary amine type, in an amount at least equal to a 50% molar amount relatively to the amount of engaged NCA, provides yields of only about 60%, provided that the solvents are dried on a 4 Å molecular sieve and that one operates between −20 and −15° C.